PROJECT SUMMARY Bacterial pathogens remain a major global health concern with associated high morbidity and mortality rates. Within host?pathogen interactions, virulence is governed by biomolecules produced by the pathogen that target different tissues during infection. Identification of effector proteins with cell-type specificity would be paramount in understanding the pathophysiology associated with infection. However, the identification of relevant protein effectors, particularly at the protein-level, has proven to be technically challenging. We sought to overcome the hurdles associated with direct identification of protein effectors by interfacing multiplexed quantitative proteomics with cell-type specific affinity capture to yield an enrichment workflow, termed Biomimetic Virulomics (BV) [Lapek et al., 2017; ACS Nano]. Our published and new preliminary data demonstrate that the pairing of biomimetics and quantitative proteomics is a powerful avenue to capture key protein effectors involved in virulence. Based on our current data we hypothesize that: 1) BV is a tool that can define host cell-type specificity of bacterial proteins; 2) the BV platform is amenable to an in vivo mouse model of infection; 3) cell-type specificity can be used as a ranking measure to target proteins for study. Herein, we will apply BV to study the proteome of the important human pathogen, Staphylococcus aureus. In Aim 1, we will define the segments of the S. aureus proteome with specificity towards human skin cells. Microbiology techniques will then be used to functionally study a subset of the BV-captured proteins in vitro. This aim is significant given S. aureus remains the leading cause of skin and soft tissue infections in the US. In Aim 2, we will seed BV to an in vivo system in order to capture S. aureus protein effector with specificity towards macrophages in a live mouse. Traditionally, in vivo, identification of bacterial proteomes in the host background has proven to be difficult. Thus, the use of BV to capture and identify bacterial-derived proteins effectors in vivo is highly innovative. Notably, this proposal will focus on characterizing proteins of unknown function. How can we expect to fully understand S aureus infection biology if nearly half of the proteins encoded in its genome are of unknown function? This proposal will be a step forward in filling this critical gap in knowledge. The scientific premise of this study is based on a powerful tool for capturing protein effector with host cell specificity directly at the protein level, a notion that was highlighted in a perspective independently written on the BV platform [Distler et al., 2017; ACS Nano]. Together, this transformative work will serve as a powerful resource and hypothesis-generating tool for host-pathogen studies from an as-yet unattained host cell-specific perspective.